Compositions and methods for the treatment of fatty acid metabolism disorders

ABSTRACT

Compositions and methods for inhibiting, treating, and/or preventing fatty acid metabolism disorders, particularly fatty acid oxidation disorders, in a subject are provided.

This application is a § 371 application of PCT/US2014/048564, filed Jul.29, 2014, which claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Patent Application No. 61/860,430, filed Jul. 31, 2013. Theforegoing applications are incorporated by reference herein.

This invention was made with government support under Grant No. 5R01HL54926-16 awarded by the National Heart, Lung, and Blood Institute. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for treating,preventing, and/or inhibiting fatty acid oxidation disorders.

BACKGROUND OF THE INVENTION

Several publications and patent documents are cited throughout thespecification in order to describe the state of the art to which thisinvention pertains. Each of these citations is incorporated herein byreference as though set forth in full.

Very long chain acyl CoA dehydrogenase (VLCAD) is one of the fouracyl-CoA dehydrogenases. VLCAD is a homodemeric mitochondrial proteinthat catalyzes the first step in the β-oxidation of fatty acids. VLCADhas activity mainly toward CoA-esters of fatty acids with 16-24 carbonsin length and is responsible for more that 80% of palmitoyl-CoAdehydrogenation in human tissues and mammalian organs and cells,indicating that it is the major contributor in mitochondrial fatty acidoxidation.

VLCAD deficiency is an autosomal recessive genetic disorder firstidentified in 1993 and now considered as the second most commonmitochondrial β-oxidation disorder. The associated disease presents withthree main phenotypes. The most severe form of VLCAD deficiency presentswith neonatal cardiomyopathy and hepatic failure and is generally fatalin the first year of life. An infantile phenotype typically presentsduring early childhood with hypoketotic hypoglycemia and hepatomegalywithout cardiomyopathy. The mildest phenotype is associated with lateronset episodic myopathic form with intermittent rhabdomyolysis, musclecramps and/or pain and exercise intolerance. To date, more than 100pathologic mutations are known including null (typically associated withthe most severe form of the disease) as well missense mutations thatoccur throughout the VLCAD protein and are associated with the milderforms of the disease. Missense mutations result in reduced enzymaticactivity and/or reduced stability of the protein leading to lower steadystate levels of acyl-CoA activity in mitochondria. Upon diagnosis ofVLCAD disease the effort is placed on the prevention of itsmanifestations. Individuals are typically placed on a low-fat formulawith supplemental calories provided through medium-chain triglycerides.Superior methods of treatment and prevention are needed.

SUMMARY OF THE INVENTION

In accordance with the instant invention, methods for treating,inhibiting, and/or preventing a fatty acid metabolism disorder,particularly a fatty acid oxidation disorder, are provided. The methodcomprises administering at least one S-nitrosylating agent to thesubject. In a particular embodiment, the fatty acid oxidation disorderis very long-chain acyl-coenzyme A dehydrogenase deficiency (VLCADD). Ina particular embodiment, the S-nitrosylating agent isS-nitroso-N-acetyl-cysteine (SNO-NAC). The methods may further compriseadministering at least one other therapeutic agent for the treatment ofthe fatty acid metabolism disorder, such as triheptanoin or bezafibrate.The methods may also comprise diagnosing a fatty acid oxidation disorderin the subject prior to administration of the S-nitrosylating agent.

In accordance with another aspect of the instant invention, compositionsfor treating, inhibiting, and/or preventing a fatty acid metabolismdisorder, particularly a fatty acid oxidation disorder, are provided. Ina particular embodiment, the composition comprises at least oneS-nitrosylating agent, at least one pharmaceutically acceptable carrier,and, optionally, at least one other therapeutic agent for the treatmentof a fatty acid metabolism disorder.

BRIEF DESCRIPTION OF THE DRAWING

FIGS. 1A-1D show the identification of the S-nitrosylation site inVLCAD. FIGS. 1A and 1B provide representative mass spectrometry (MS)spectra of doubly-charged sulfonic acid-containing tryptic peptide,Ser²³¹-Ser-Ala-Ile-Pro-Ser-Pro-Cys²³⁸-Gly-Lys-Tyr-Tyr-Thr-Leu-Asn-Gly-Ser-Lys²⁴⁸(SEQ ID NO: 3; monoisotopic m/z=960.9539 and 960.9561) from very longchain specific acyl dehydrogenase (VLCAD) acquired in liver homogenatesfrom ob/ob GSNO injected mice and eNOS^(−/−) liver after ex vivotreatment with GSNO (N=3 biological replicates). Spectra for the samepeptide were acquired in wild-type mouse liver. FIGS. 1C and 1D showthat MS/MS spectra confirmed the sequence and site of sulfonic acidcontaining peptide from VLCAD identified in ob/ob mouse liver injectedwith GSNO and eNOS^(−/−) liver after ex vivo treatment with GSNO (N=3biological replicates). SEQ ID NO: 3 is shown in FIGS. 1C and 1D.

FIG. 2A provides a graph of the rate of ³H-labeled palmitoyl-CoAoxidation in liver homogenates from wild-type and ob/ob mice. *P<0.05 byanalysis of variance (ANOVA) with Bonferroni post hoc test betweenwild-type and ob/ob PBS-treated mice (n=4 mice). **P<0.01 by ANOVA withBonferroni post hoc test between ob/ob PBS- and ob/ob GSNO-treated mice(n=4 mice). FIG. 2B shows liver triglyceride measurements in wild-typemice, PBS-treated ob/ob mice, and GSNO-treated ob/ob mice. *P<0.0001 byANOVA with Bonferroni post hoc test between wild-type and ob/obPBS-treated mice (n=4 mice). **P<0.005 by ANOVA with Bonferroni post hoctest between ob/ob PBS- and ob/ob GSNO-treated mice (n=4 mice). FIG. 2Cshows serum triglyceride measurements in wild-type mice, PBS-treatedob/ob mice, and GSNO-treated ob/ob mice. No statistical difference (n=4mice). FIG. 2D provides representative measurements of VLCAD initialvelocity measured as a function of palmitoyl-CoA concentration in liverhomogenates from wild-type, ob/ob PBS, and ob/ob GSNO mice. FIGS. 2E and2F provides kinetic analysis of VLCAD enzymatic activity reveals similarV_(max) but significantly higher KM in PBS-treated ob/ob mouse liver ascompared to wild-type and GSNO-treated ob/ob mouse liver. *P<0.05 byANOVA with Bonferroni post hoc test (n=3 biological replicates). FIG. 2Gprovides images of trichrome staining of liver tissue showing diminishedfatty acid deposition in ob/ob mice treated with GSNO (bottom) ascompared to PBS-treated ob/ob mice (top). Images are representative offour different mouse livers examined. The scale bar corresponds to 15 μmfor left and to 7.5 μm for right. FIG. 2H shows VLCAD specific activitydetermined by monitoring palmitoyl-CoA (0.25 mM) mediated reduction offerricenium ion in liver homogenates from wild type and eNOS^(−/−)mouse. The specific activity of eNOS^(−/−) was significantly lower thanwild-type or eNOS^(−/−) liver tissue treated with 5 μM GSNO 30 minutesprior to the assay. *p<0.0001, **p<0.05 by ANOVA with Bonfferoni's posthoc test. N=3 biological replicates. FIG. 2I shows Western blot analysisof VLCAD fractions that did not bind (U=unmodified VLCAD) and bound toorganomercury (B=S-nitrosylated VLCAD) from eNOS^(−/−) mouse liverhomogenates and eNOS^(−/−) mouse liver homogenates treated with GSNO.The absence of VLCAD from the bound fraction of the untreated homogenateindicated that the protein was not S-nitrosylated in eNOS^(−/−) liver.Ex vivo treatment with GSNO resulted in S-nitrosylation of a fraction ofthe protein as it was indicated by the immunoreactivity in the boundfraction of the treated homogenate. This experiment was repeated twicemore with similar results. FIG. 2J shows typical tracings of VLCADacyl-dehydrogenase activity using 150 μM ferricenium hexafluorophosphateas electron acceptor. The activity was measured in liver homogenatesfrom GSNO-injected ob/ob mice after the addition of 0.125 mMpalmitoyl-CoA. The specificity of the assay for measuring VLCAD activitywas confirmed by the abolishment of ferricenium reduction in thepresence of anti-VLCAD antibodies. The effect of S-nitrosylation onVLCAD activity was confirmed by the loss of enzymatic activity afterUV-photolysis of liver lysate. This experiment was repeated once more inanother biological replicate with identical results. FIG. 2K presentsthe initial velocity (V₀) measured as a function of palmitoyl-CoAconcentration in liver homogenates from three groups of mice. *p<0.05 byt-test. N=3 biological replicates.

FIG. 3A provides representative Western blots assessing VLCAD in nativegel (top panel) and in SDS gel (middle panel). Noncontiguous lanes froma single experiment are indicated by black lines. FIG. 3B shows thequantification of abundance of VLCAD in SDS gels under reducingconditions in total liver homogenates and enriched mitochondriafractions from liver. No statistical difference by ANOVA. n=3 differentmice. cyt c, cytochrome c. FIG. 3C provides a representative Westernblot for VLCAD in liver homogenates eluted from organomercury resin. Thesignal intensity was used to determine the abundance of S-nitrosylatedVLCAD in the bound (B) fraction and the unmodified VLCAD present in theunbound (U) fraction. The data were repeated in two independent liverhomogenates.

FIG. 4A provides representative Western blot analysis of unbound (VLCAD)and bound fractions (SNO-VLCAD) collected after mercury-assisted capturein cell lysates. Hepa 1 to 6 cells transiently expressing eitherFLAG®-tagged wild-type or C238A VLCAD were exposed to GSNO. The unboundfraction indicates the abundance of the unmodified protein. The boundfraction indicates the abundance of S-nitrosylated VLCAD. The abundanceof both was determined by using a calibrated antibody binding curveusing purified VLCAD. The fraction of S-nitrosylated VLCAD as percentageof VLCAD is indicated. ND, not detected. n=3 biological replicates.Noncontiguous lanes from a single experiment are indicated by blacklines. FIG. 4B shows the specific activity of VLCAD was significantlyhigher in GSNO-treated cells expressing wild-type VLCAD but not inGSNO-treated cells expressing equivalent amount of C238A VLCAD mutantprotein. *P<0.001, **P<0.05 by ANOVA with Bonferroni post hoc test. n=3biological replicates.

FIG. 5A provides the crystal structure of VLCAD dimer with the site ofS-nitrosylation on Cys²³⁸ annotated in gold on both monomers. Note thatCys²³⁸ is in a loop, a secondary conformation that typically showsincreased flexibility. FIG. 5B shows higher frequency mode indicatesextensive movement of Cys²³⁸ upon S-nitrosylation. Normal mode analysisof unmodified and SNO-VLCAD using the web interface ElNémo(igs-server.cnrs-mrs.fr/elnemo/index.html). The bottom line depicts theR² values for the amino acid residues of the unmodified VLCAD whereasthe top line depicts the values for the same residues of theS-nitrosylated VLCAD. Note the higher R² value for the S-nitrosocysteine238 compared to the unmodified cysteine indicative of the enhancedmovement of Cys²³⁸ upon S-nitrosylation.

FIGS. 6A and 6B provide graphs of the VLCAD kinetic parameters of celllines 1 and 2, respectively. Five μg of cell lysate were mixed with 150μM ferrocenium followed by the addition of the indicated concentrationsof palmitoyl-CoA. The decrease in ferricenium absorbance as a functionof time at 300 nm was recorded and the initial velocity of the enzymewas determined from the slope of the curve from time 0 to the time thatcorresponded to 5% of total change of absorbance.

DETAILED DESCRIPTION OF THE INVENTION

Protein S-nitrosylation is a major nitric oxide-derived reversibleposttranslational modification that regulates enzymatic activity,protein localization, and stability, and that also plays a role innitric oxide-mediated signaling (Stamler et al. (2001) Cell 106:675-683;Hess et al. (2005) Nat. Rev. Mol. Cell Biol., 6:150-166; Benhar et al.(2008) Science 320:1050-1054; Jaffrey et al. (2001) Nat. Cell Biol.,3:193-197; Kornberg et al. (2010) Nat. Cell Biol., 12:1094-1100;Matsushita et al. (2003) Cell 115:139-150; Mitchell et al. (2005) Nat.Chem. Biol., 1:154-158; Cho et al. (2009) Science 324:102-105; Mannicket al. (2001) J. Cell Biol., 154:1111-1116). Although functional rolesfor S-nitrosylation have been documented for individual proteins, aglobal analysis of S-nitrosylation and the S-nitrosylation sites underphysiological conditions in vivo remains limited. To this end, a massspectrometry (MS)-based proteomic approach has been implemented whichallows for the site-specific identification of S-nitrosocysteineresidues in complex mixtures (Doulias et al. (2010) Proc. Natl. Acad.Sci., 107:16958-16963). The method is based on selective enrichmenteither of S-nitrosocysteine peptides or intact S-nitrosylated proteinswith organomercury compounds. The peptides are released with performicacid, which oxidizes the cysteine to sulfonic acid, enabling precisedetection of the modified peptides by MS. Alternatively, proteins can beeluted intact and probed with antibodies against specific proteins,enabling quantification of the modified protein molecules. To ensurespecificity of detection, negative controls are generated bypretreatment of samples with ultraviolet (UV) light, which eliminatesS-nitrosocysteine, and analyzed under the same conditions (Doulias etal. (2010) Proc. Natl. Acad. Sci., 107:16958-16963). These methodologieswere used to identify endogenous S-nitrosylated proteins in sixwild-type mouse organs, as well as in the same tissues from mice lackingthe endothelial nitric oxide synthase (eNOS). A global discovery of theS-nitrosocysteine proteome in mice reveals potential functionalregulation of core biochemical pathways by S-nitrosylation and thechanges in this proteome in the absence of one of the major enzymaticsources of nitric oxide. Indeed, selective S-nitrosylation of enzymesparticipating in glycolysis, gluconeogenesis, tricarboxylic acid cycle,and oxidative phosphorylation has been found, indicating that thispost-translational modification regulates metabolism and mitochondrialbioenergetics. S-nitrosylation of the murine liver enzyme very longchain acyl-coenzymeA (CoA) dehydrogenase (VLCAD) at Cys²³⁸, which wasabsent in mice lacking endothelial nitric oxide synthase, improved itscatalytic efficiency. Moreover, the administration of an S-nitrosylatingagent to cells with a mutant VLCAD resulted in increased enzymaticactivity. These data implicate protein S-nitrosylation in the regulationof β-oxidation of fatty acids in mitochondria.

The instant invention encompasses methods of inhibiting, treating,and/or preventing a fatty acid metabolism disorder. In a particularembodiment, the fatty acid metabolism disorder is a fatty acid oxidationdisorder. Fatty acid oxidation disorders include, without limitation,very long-chain acyl-coenzyme A dehydrogenase deficiency (VLCADD; e.g.,16-24 carbons), long-chain acyl-coenzyme A dehydrogenase deficiency(LCADD; e.g., 12-18 carbons), long-chain 3-hydroxyacyl-coenzyme Adehydrogenase deficiency (LCHADD), medium-chain acyl-CoA dehydrogenasedeficiency (MCADD; e.g., 6-12 carbons), short chain acyl-CoAdehydrogenase deficiency (SCADD; e.g., 4-6 carbons), medium/short chainL-3-hydroxyacyl-CoA dehydrogenase deficiency (M/SCHADD), multipleacyl-CoA dehydrogenase deficiency (MADD), mitochondrial trifunctionalprotein deficiency, short chain 3-ketoacyl-CoA thiolase deficiency(SKATD), medium chain 3-ketoacyl-CoA thiolase deficiency (MCKATD),2,4-dienoyl-CoA reductase deficiency, and glutaric acidemia type II(GA-II). In a particular embodiment, the fatty acid oxidation disorderis VLCADD.

The methods of the instant invention comprise administering at least onenitrosylating agent, particularly an S-nitrosylating agent, to asubject. As used herein the term “nitrosylation” refers to the additionof nitric oxide (NO) to a polypeptide, particularly at a thiol group(SH), oxygen, carbon or nitrogen. The term “S-nitrosylation” refers tothe addition of a nitric oxide moiety to a thiol group, thereby formingan S-nitrosothiol (SNO). An “S-nitrosylating agent” refers to a compoundthat transfers a nitric oxide group to the thiol of a polypeptide toform an S-nitrosothiol. Examples of S-nitrosylating agents are providedin Feelisch, J., Stamler, J. S. (1996) Donors of Nitrogen Oxides.Feelisch, M. Stamler, J. S. eds. Methods in Nitric Oxide Research, JohnWiley & Sons, Ltd. Chichester, UK. S-nitrosylating agents include,without limitation, S-nitrosylating agents that directly nitrosylate(e.g., GSNO), precursor agents that are modified in vivo toS-nitrosylating agents (e.g., N-acetyl cysteine (NAC)), and nitric oxidegenerators that produce nitrosylators (e.g., nitrite plus GSH or NAC),as well as esters and/or salts thereof. S-nitrosylating agents include,without limitation, acidic nitrite, nitrosyl chloride, alkyl nitrate(e.g., ethyl nitrite), amyl nitrite, glutathione (GSH), glutathioneoligomer, S-nitrosoglutathione (GSNO), S-nitrosocysteinyl glycine,S-nitrosocysteine, N-acetyl cysteine, S-nitroso-N-acetyl cysteine,nitroglycerine, nitroprusside, nitric oxide, S-nitrosohemoglobin,S-nitrosoalbumin, 5-nitroso-N-acetylpenicillamine,S-nitroso-gamma-methyl-L-homocysteine, 5-nitroso-L-homocysteine,S-nitroso-gamma-thio-L-leucine, S-nitroso-delta-thio-L-leucine, andS-nitrosoalbumin, as well as pharmaceutically acceptable salts thereof.In a particular embodiment, the S-nitrosylating agent isS-nitroso-N-acetyl-cysteine (SNO-NAC). The methods of the instantinvention may further comprise the administration (sequentially and/orsimultaneously) of at least one other therapeutic for the treatment ofthe fatty acid metabolism disorder. For example, the methods may furthercomprise administering triheptanoin and/or bezafibrate. The subject mayalso be administered a diet that reduces or eliminates the presence ofproblematic fatty acids (e.g., fatty acids that are substrates for thedeficient enzyme), e.g., fatty acids with 12 or fewer carbons or fattyacids with more than 12 or 16 carbons (e.g., for VLCADD).

The S-nitrosylating agent may be delivered to the subject as acomposition with at least one pharmaceutically acceptable carrier. In aparticular embodiment, the composition further comprises at least oneother therapeutic agent for the treatment of the fatty acid metabolismdisorder, as described above.

As stated hereinabove, the instant invention encompasses methods ofinhibiting, treating, and/or preventing a fatty acid metabolism disorderin a subject. The method may further comprise diagnosing a fatty acidmetabolism disorder in the subject prior to administration of thetherapeutic agents of the instant invention. More specifically, themethod may further comprise determining whether the subject hasdeficient VLCAD enzymatic activity compared to healthy subjects. Methodsof determining VLCAD activity are known in the art. For example, abiological sample may be obtained from the subject and a VLCAD enzymaticassay may be performed (e.g., an acyl-CoA dehydrogenase assay usingferricenium ion) and compared to a standard value (e.g., from healthysubjects and/or a subject with the fatty acid metabolism disorder) ordirectly compared to a biological sample from a healthy subject and/or asubject with the fatty acid metabolism disorder. Alternatively (oradditionally), VLCAD activity may be measured by determining whether thesubject has a missense mutation in VLCAD (e.g., those presented inTable 1) and/or null mutation (e.g., resulting in a VLCAD with deficientactivity). VLCAD mutations are also provided in Gobin-Limballe et al.(Am. J. Hum. Genet. (2007) 81:1133-1143 (see, e.g., FIG. 1 and Table 1))and Andresen et al. (Am. J. Hum. Genet. (1999) 64:479-494 (see, e.g.,Table 2)). Each of these references is incorporated herein by referenceas though set forth in full. Methods of detecting VLCAD mutations areknown in the art. For example, a biological sample may be obtained fromthe subject and the VLCAD gene may be sequenced or the nucleic acidsfrom the biological sample may be contacted with one or more probes(e.g., specific for wild-type or a mutant). Alternatively, the presenceof mutant VLCAD may be detected through the use of antibodiesimmunologically specific for wild-type or mutant VLCAD. When the subjectis determined to have deficient VLCAD enzymatic activity and/ordetermined to carry a missense or null mutation, the subject may then betreated by the methods of the instant invention.

The compositions of the present invention can be administered by anysuitable route, for example, by injection (e.g., for local, direct, orsystemic administration), oral, pulmonary, topical, nasal or other modesof administration. The composition may be administered by any suitablemeans, including parenteral, intramuscular, intravenous, intraarterial,intraperitoneal, subcutaneous, topical, inhalatory, transdermal,intrapulmonary, intraareterial, intrarectal, intramuscular, andintranasal administration. In a particular embodiment, the compositionis administered orally and/or intraperitoneally. In general, thepharmaceutically acceptable carrier of the composition is selected fromthe group of diluents, preservatives, solubilizers, emulsifiers,adjuvants and/or carriers. The compositions can include diluents ofvarious buffer content (e.g., Tris HCl, acetate, phosphate), pH andionic strength; and additives such as detergents and solubilizing agents(e.g., Tween 80, Polysorbate 80), anti oxidants (e.g., ascorbic acid,sodium metabisulfite), preservatives (e.g., Thimersol, benzyl alcohol)and bulking substances (e.g., lactose, mannitol). The compositions canalso be incorporated into particulate preparations of polymericcompounds such as polyesters, polyamino acids, hydrogels,polylactide/glycolide copolymers, ethylenevinylacetate copolymers,polylactic acid, polyglycolic acid, etc., or into liposomes. Suchcompositions may influence the physical state, stability, rate of invivo release, and rate of in vivo clearance of components of apharmaceutical composition of the present invention (see, e.g.,Remington's Pharmaceutical Sciences and Remington: The Science andPractice of Pharmacy). The pharmaceutical composition of the presentinvention can be prepared, for example, in liquid form, or can be indried powder form (e.g., lyophilized for later reconstitution).

The therapeutic agents described herein will generally be administeredto a subject/patient as a pharmaceutical preparation. The term “patient”as used herein refers to human or animal subjects. The compositions ofthe instant invention may be employed therapeutically orprophylactically, under the guidance of a physician.

The compositions comprising the agent of the instant invention may beconveniently formulated for administration with any pharmaceuticallyacceptable carrier(s). The concentration of agent in the chosen mediummay be varied and the medium may be chosen based on the desired route ofadministration of the pharmaceutical preparation. Except insofar as anyconventional media or agent is incompatible with the agent to beadministered, its use in the pharmaceutical preparation is contemplated.

The dose and dosage regimen of the agent according to the invention thatis suitable for administration to a particular patient may be determinedby a physician considering the patient's age, sex, weight, generalmedical condition, and the specific condition for which the agent isbeing administered to be treated or prevented and the severity thereof.The physician may also take into account the route of administration,the pharmaceutical carrier, and the agent's biological activity.Selection of a suitable pharmaceutical preparation will also depend uponthe mode of administration chosen.

A pharmaceutical preparation of the invention may be formulated indosage unit form for ease of administration and uniformity of dosage.Dosage unit form, as used herein, refers to a physically discrete unitof the pharmaceutical preparation appropriate for the patient undergoingtreatment or prevention therapy. Each dosage should contain a quantityof active ingredient calculated to produce the desired effect inassociation with the selected pharmaceutical carrier. Procedures fordetermining the appropriate dosage unit are well known to those skilledin the art.

Dosage units may be proportionately increased or decreased based on theweight of the patient. Appropriate concentrations for alleviation orprevention of a particular condition may be determined by dosageconcentration curve calculations, as known in the art.

The pharmaceutical preparation comprising the agent may be administeredat appropriate intervals, for example, at least twice a day or moreuntil the pathological symptoms are reduced or alleviated, after whichthe dosage may be reduced to a maintenance level. The appropriateinterval in a particular case would normally depend on the condition ofthe patient.

Toxicity and efficacy (e.g., therapeutic, preventative) of theparticular formulas described herein can be determined by standardpharmaceutical procedures such as, without limitation, in vitro, in cellcultures, ex vivo, or on experimental animals. The data obtained fromthese studies can be used in formulating a range of dosage for use inhuman. The dosage may vary depending upon form and route ofadministration. Dosage amount and interval may be adjusted individuallyto levels of the active ingredient which are sufficient to deliver atherapeutically or prophylactically effective amount.

Definitions

The following definitions are provided to facilitate an understanding ofthe present invention:

The singular forms “a,” “an,” and “the” include plural referents unlessthe context clearly dictates otherwise.

“Pharmaceutically acceptable” indicates approval by a regulatory agencyof the Federal or a state government or listed in the U.S. Pharmacopeiaor other generally recognized pharmacopeia for use in animals, and moreparticularly in humans.

A “carrier” refers to, for example, a diluent, adjuvant, preservative(e.g., Thimersol, benzyl alcohol), anti-oxidant (e.g., ascorbic acid,sodium metabisulfite), solubilizer (e.g., Tween® 80, Polysorbate 80),emulsifier, buffer (e.g., Tris HCl, acetate, phosphate), antimicrobial,bulking substance (e.g., lactose, mannitol), excipient, auxiliary agentor vehicle with which an active agent of the present invention isadministered. Pharmaceutically acceptable carriers can be sterileliquids, such as water and oils, including those of petroleum, animal,vegetable or synthetic origin. Water or aqueous saline solutions andaqueous dextrose and glycerol solutions may be employed as carriers,particularly for injectable solutions. Suitable pharmaceutical carriersare described in “Remington's Pharmaceutical Sciences” by E. W. Martin(Mack Publishing Co., Easton, Pa.); Gennaro, A. R., Remington: TheScience and Practice of Pharmacy, (Lippincott, Williams and Wilkins);Liberman, et al., Eds., Pharmaceutical Dosage Forms, Marcel Decker, NewYork, N.Y.; and Kibbe, et al., Eds., Handbook of PharmaceuticalExcipients, American Pharmaceutical Association, Washington.

The term “treat” as used herein refers to any type of treatment thatimparts a benefit to a patient afflicted with a disease, includingimprovement in the condition of the patient (e.g., in one or moresymptoms), delay in the progression of the condition, etc.

As used herein, the term “prevent” refers to the prophylactic treatmentof a subject who is at risk of developing a condition (e.g., fatty acidoxidation disorder) resulting in a decrease in the probability that thesubject will develop the condition.

A “therapeutically effective amount” of a compound or a pharmaceuticalcomposition refers to an amount effective to prevent, inhibit, or treata particular disorder or disease and/or the symptoms thereof. Forexample, “therapeutically effective amount” may refer to an amountsufficient to modulate fatty acid oxidation in a subject.

As used herein, the term “subject” refers to an animal, particularly amammal, particularly a human.

As used herein, a “biological sample” refers to a sample of biologicalmaterial obtained from a subject, particularly a human subject,including a tissue, a tissue sample, cell(s), and a biological fluid(e.g., blood).

The term “probe” as used herein refers to an oligonucleotide,polynucleotide or nucleic acid, either RNA or DNA, whether occurringnaturally as in a purified restriction enzyme digest or producedsynthetically, which is capable of annealing with or specificallyhybridizing to a nucleic acid with sequences complementary to the probe.A probe may be either single-stranded or double-stranded. The exactlength of the probe will depend upon many factors, includingtemperature, source of probe and use of the method. For example, fordiagnostic applications, depending on the complexity of the targetsequence, the oligonucleotide probe typically contains 15-25 or morenucleotides, although it may contain fewer nucleotides. The probesherein are selected to be complementary to different strands of aparticular target nucleic acid sequence. This means that the probes mustbe sufficiently complementary so as to be able to “specificallyhybridize” or anneal with their respective target strands under a set ofpre-determined conditions. Therefore, the probe sequence need notreflect the exact complementary sequence of the target. For example, anon-complementary nucleotide fragment may be attached to the 5′ or 3′end of the probe, with the remainder of the probe sequence beingcomplementary to the target strand. Alternatively, non-complementarybases or longer sequences can be interspersed into the probe, providedthat the probe sequence has sufficient complementarity with the sequenceof the target nucleic acid to anneal therewith specifically.

The following examples are provided to illustrate various embodiments ofthe present invention. They are not intended to limit the invention inany way.

Example 1 Materials and Methods

Chemicals and Reagents

Palmitoyl-CoA lithium salt and ferricenium hexafluorophosphate wereobtained from Sigma-Aldrich. Mouse monoclonal antibodies against FLAG®,glyceraldehyde-3-phosphate dehydrogenase, and cytochrome c oxidasesubunit I were from Stratagene and Abcam, respectively. Rabbit and goat(G-16 clone) polyclonal antibodies against VLCAD were obtained fromGeneTex and Santa Cruz Biotechnology, respectively. All chemicals andreagents used were of analytical grade.

Isolation of Mouse Organs and Preparation of Protein Homogenates

All mouse studies were reviewed and approved by the Institutional AnimalCare and Use Committee of the Children's Hospital of PhiladelphiaResearch Institute. Wild-type C57BL/6J, Nos3^(tm/Unc) (eNOS^(−/−))C57BL/6J, and Lep^(ob) (ob/ob) C57BL/6J adult mice were obtained fromJackson Laboratories. Food intake and bodyweight were recorded for ob/obmice during the course of 4 weeks when the mice were being injected withPBS or 5 mM GSNO every 2 days. The average food intake was 42±9 and41±10 g for PBS- and GSNO-injected ob/ob mice, respectively (n=4 miceper genotype). The average bodyweight change for the same period of timewas 28.2±1.0 and 28.1±4.0 for ob/ob PBS- and ob/ob GSNO-injected mice,respectively (n=4 mice per genotype). Mice were anesthetized by CO₂, andblood was collected before being perfused through the left ventricle.Intact organs were collected, immediately frozen in liquid nitrogen, andstored at −80° C. until use. Tissues were homogenized into 3 ml of lysisbuffer [250 mM Hepes-NaOH (pH7.7) containing 1 mM diethylenetriaminepentaacetic acid, 0.1 mM neocuproine, 1% Triton X-100, and proteaseinhibitors] on ice with a Teflon pestle and a Jumbo Stirrer (FisherScientific). The homogenates were then centrifuged at 13,000 g for 30minutes at 4° C. The soluble protein fraction was collected, and theprotein concentration was determined by the Bradford assay. Samplepreparation and generation of the negative control samples wereperformed as described (Doulias et al. (2010) Proc. Natl. Acad. Sci.,107:16958-16963).

Chemical Enrichment and Site-Specific Identification of theS-Nitrosocysteine Proteome

A detailed experimental procedure for the preparation and activation ofcolumns, homogenate preparation for reaction with organic mercury resin,has been described (Doulias et al. (2010) Proc. Natl. Acad. Sci.,107:16958-16963). Three biological replicates from each organ wereanalyzed. Each sample had a corresponding UV-pretreated negative controlanalyzed under identical conditions. The false identification rate was<6% for brain and <3% for all other organs. For washes, stringentconditions were selected due to the differences in lipid content amongthe six organs. Columns were initially washed with 50 bed volumes of 50mM tris-HCl (pH 7.4) containing 300 mM NaCl and 0.5% SDS, followed by 50bed volumes of the same buffer containing 0.05% SDS. Columns were washedwith 50 bed volumes of 50 mM tris-HCl containing 300 mM NaCl (pH 7.4),1% Triton X-100, and 1 M urea, followed by 50 bed volumes of the samebuffer containing 0.1% Triton X-100 and 0.1 M urea. Finally, columnswere washed with 200 bed volumes of water before proteins were elutedwith 10 ml of 50 mM β-mercaptoethanol in water. Samples wereconcentrated and analyzed by gel-LC (liquid chromatography)-MS/MSanalysis. For on-column digestion after the final wash with water, thecolumns were washed with 10 bed volumes of 0.1 M ammonium bicarbonate.Bound proteins were subjected to digestion by the addition of TrypsinGold (1 μg/ml) (Promega) in one bed volume of 0.1 M ammonium in the darkfor 16 hours at room temperature. The resin was next washed with 40 bedvolumes of 1M ammonium bicarbonate (pH 7.4) containing 300 mM NaCl,followed by 40 volumes of the same buffer without NaCl. Columns werethen washed with 40 volumes of 0.1 M ammonium bicarbonate followed by200 volumes of deionized water. To elute bound peptides, the resin wasincubated with one bed volume of performic acid in water (performic acidis synthesized by reacting 1% formic acid and 0.5% hydrogen peroxide forat least 60 minutes at room temperature with rocking in a glass vialshielded from light) for 30 minutes at room temperature (Doulias et al.(2010) Proc. Natl. Acad. Sci., 107:16958-16963). Eluted peptides wererecovered by washing the resin with one bed volume of deionized water.Eluates were stored at −80° C. overnight followed by lyophilization andresuspension into 300 μl of 0.1% formic acid. Peptide suspensions weretransferred to low-retention tubes (Axygen), and the volume was reducedto 30 μl by speed vacuum. Twenty microliters of peptide suspension wastransferred to a high-performance liquid chromatography vial andsubmitted for LC-MS/MS analysis.

The details for in-gel digestion and the conditions of MS/MS analysishave been provided previously (Doulias et al. (2010) Proc. Natl. Acad.Sci., 107:16958-16963; Keene et al. (2009) Proteomics 9:768-782).Post-MS analysis to generate the S-nitrosocysteine proteomes (Tables S1to S6) has been performed as described with the following exception:Cysteine containing peptides that were not matched to proteins from thesame organ were matched with proteins identified independently in otherorgans.

Subcellular localization was determined by either existing UniProtannotation (www.uniprot.org) or prediction by BaCelLo(gper.biocomp.unibo.it/bacello). Functional analysis to identify thebiological functions that were most important was performed with UniProtand Fatigo (www.fatigo.org). The raw MS/MS data are deposited atwww.research.chop.edu/tools/msms/spectra.pdf.

VLCAD Activity Assay

VLCAD enzymatic activity was assessed as described (Lehman et al. (1990)Anal. Biochem., 186:280-284). Briefly, ferricenium ion was used as anartificial electron acceptor for VLCAD-mediated palmitoyl-CoAdehydrogenation. Liver homogenate (final concentration of 0.03 μg/μl) orcell lysate (final concentration of 0.09 μg/μl) in 100 mM potassiumphosphate buffer (pH 7.2) containing 0.2% Triton X-100 and 0.1 mM EDTAwas mixed with 150 mM ferricenium ion, and the reaction was initiated bythe addition of palmitoyl-CoA (the final volume of the assay was 130μl). The decrease in ferricenium absorbance as a function of time at 300nm was recorded, and the initial velocity (V_(o)) of the enzyme wasdetermined from the slope of the curve from time 0 (when palmitoyl-CoAwas added) to the time that corresponded to 5% of total change ofabsorbance. At least nine concentrations of palmitoyl-CoA, ranging from0.015 to 2 mM, were used for the experimental determination of theapparent V_(max) and K_(M) of VLCAD for each animal. The experimentaldata were fitted to nonlinear regression to the Michaelis-Mentenequation in GraphPad Prism software. A unit of enzyme activity isdefined as the amount of enzyme that causes the reduction of 1 mmol offerricenium per minute at room temperature (ε=4.3 mM⁻¹ cm⁻¹ at 300 nm)(Izai et al. (1992) J. Biol. Chem., 267:1027-1033). For the experimentsto test the specificity of the assay, 10 mg of anti-VLCAD antibodies waspreincubated with liver homogenate for 20 minutes. When UV photolysiswas used to eliminate S-nitrosocysteines, the liver homogenate wasilluminated under a conventional UV transilluminator for 10 minutes onice. The measurement of specific activity of VLCAD in liver homogenates(FIG. 2H) and cell lysates (FIG. 4) was performed in the presence of0.25 and 0.125 mM palmitoyl-CoA, respectively.

To quantify the fraction of S-nitrosylated VLCAD, the protein wascaptured onto the organomercury resin. After extensive washing, thebound proteins were eluted and probed with antibodies against VLCAD.After extensive washing, the bound proteins were eluted and probed withantibodies against VLCAD.

Liver Histology and Quantification of Triglyceride Concentrations

Formalin-fixed liver sections were stained with Trichrome Stain Kit(Sigma) according to the manufacturer's instructions. Triglycerides wereextracted from liver according to the Folch method (Folch et al. (1957)J. Biol. Chem., 226:497-509). Serum and liver triglycerideconcentrations were quantified with a triglyceride quantification kitfollowing the manufacturer's instructions (Abcam).

Measurement of β-Oxidation of Fatty Acids

One milligram of protein suspension was added in 1 ml of Krebs-Ringerbicarbonate buffer containing fatty acid-free bovine serum albumin (2.5mg/ml), 2.5 mM palmitic acid, 10 mM carnitine, and 4 mCi of9,10-³H-palmitoyl-CoA (Biomedicals). The mixture was rocked for 2 hoursin the dark at 37° C., followed by Folch-based separation of9,10-³H-palmitoyl-CoA and ³H₂O (Folch et al. (1957) J. Biol. Chem.,226:497-509). The aqueous phase was collected, and proteins wereprecipitated by the addition of 10% TCA followed by centrifugation at8000 g for 10 minutes at room temperature. Remaining radioactivepalmitoyl-CoA was eliminated by strong anion exchange chromatographywith AG 1-X8 formate resin (Life Science). The effluent containing the³H₂O was collected and used for scintillation counting. Each experimentwas coupled with a background measurement, a sample containing noprotein. The background count number was subtracted from eachmeasurement corresponding to analysis samples.

Site-Directed Mutagenesis to Generate the C238A VLCAD Mutant

The QuikChange® Lightning Site-Directed Mutagenesis Kit (AgilentTechnologies) was used to introduce single amino acid mutations incomplementary DNA encoding VLCAD. A cysteine-to-alanine mutant at aminoacid position 238 was generated with Ex-Mm01013-M14 (hereinafterreferred to as pVLCAD-3×FLAG®), a plasmid encoding mouse VLCAD fused tothree FLAG® tags in the C-terminal region of the protein (GeneCopoeia),as a template. The forward primer5′-TCAGCCATACCCAGCCCCGCTGGAAAATATTACACTCTC-3′ (SEQ ID NO: 1) and thereverse primer 5′-GAGAGTGTAATATTTTCCAGCGGGGCTGGGTATGGCTGA-3′ (SEQ ID NO:2) were used to substitute an alanine codon for a cysteine codon inpVLCAD-3×FLAG®, thereby generating pVLCAD-C238A-3×FLAG®. The sequence ofboth pVLCAD-3×FLAG® and pVLCAD-C238A-3×FLAG® was verified before use insubsequent experiments.

Cell Culture, Transfection, and Treatment with GSNO

Hepa 1 to 6 cells were grown in Dulbecco's modified Eagle's medium(DMEM) containing 10% fetal bovine serum, 2 mM glutamine, penicillin(100 U/ml), and streptomycin (100 ng/ml) at 37° C. in air with 5% CO₂.Cells were plated at a density of 5×10⁴ cells/cm² and cultured undernormal conditions for 24 hours. Cells were transfected with FLAG®-taggedwild-type VLCAD or FLAG®-tagged C238A VLCAD with Lipofectamine® 2000reagent (Life Technologies) according to the protocol provided by themanufacturer. Forty-eight hours after transfection, the growth mediumwas replaced with serum-free DMEM, and 250 mM freshly prepared GSNO wasadded for 30 minutes, after which the cells were extensively washed withPBS. Cell lysates were obtained immediately or 60, 120, and 240 minutesafter the removal of GSNO. The samples were protected from UV lightexposure. Lysates were assayed for protein concentration, and equalamounts of protein per sample were used for organic mercury-assistedcapture.

Structure Generation of S-Nitrosylated Cysteine and Normal Mode Analysis

The crystal structure of human VLCAD was downloaded from PDB (ID 2UXW).Disordered or missing residues were completed with the Mutagenesis toolin PyMol (www.pymol.org). S-nitrosocysteine was generated with the“S-nitrosator” Python script from the Timerghazin laboratory in theMolecular Modeling Toolkit. S-nitrosator uses the coordinates ofthioredoxin and χ³ values calculated from PCM-ONIOM(PBEO/def2-TZVPPD:AmberFF) calculation of an S-nitrosocysteine residuein an a helix content. ElNémo (Suhre et al. (2004) Nucleic Acids Res.,32:W610-W614) was used to observe the 100 lowest frequency modes, andperturbed models were generated for the first five nontrivial modes ofVLCAD. Residue mean square displacement (r²) was used to identifyprotein movement.

Statistical Analysis

Data were analyzed with GraphPad Prism 5.0d software. All normallydistributed data were displayed as means±SD. Groups were analyzed byone-way ANOVA.

Results

A Mouse S-Nitrosocysteine Proteome

In wild-type mouse brain, heart, kidney, liver, lung, and thymus, 1011S-nitrosocysteine-containing peptides were identified on 647 proteins.Extensive literature searches indicated that this expandedS-nitrosocysteine proteome identified 46 proteins previously reported tobe modified under physiological conditions and uncovered 971 previouslyunknown sites of endogenous S-nitrosylation. In all six organs, thenumber of S-nitrosylation sites exceeded the number of proteins,indicating a potential role of poly-S-nitrosylation in the regulation ofprotein function in vivo (Simon et al. (1996) Proc. Natl. Acad. Sci.,93:4736-4741). Comparison of the proteins identified in the six organsin at least three biological replicates for each organ revealed that, onaverage, 72% of the proteins were identified in more than one organ,indicating that similar patterns of S-nitrosylation serve globalfunctions in vivo. Moreover, these data indicated that the methodologiesused to acquire these proteomes were accurate and reproducible. Proteinsidentified only in one organ ranged from 21 to 46%, indicating thatS-nitrosylation can also serve organ-specific roles. The dependency ofthe sites of S-nitrosylation in vivo to nitric oxide generated by eNOSwas explored by analyzing the endogenous sites of modification in eNOSnull mice (eNOS^(−/−)) in the same organs. eNOS substantiallycontributed to the endogenous S-nitrosocysteine proteome becauseS-nitrosylation of 47 to 87% of the proteins identified required thepresence of eNOS. The brain and the heart had the lowest dependency oneNOS activity compared to the other organs, indicating the contributionof other isoforms such as neuronal nitric oxide synthase forS-nitrosylation of proteins in these organs. The absence of asubstantial number of S-nitrosocysteine peptides in organs fromeNOS^(−/−) mice reinforced the accuracy of the methodologies inidentifying this S-nitrosocysteine proteome.

An overview of the subcellular localization of the S-nitrosoproteomerevealed a tissue-wide significant enrichment for cytosolic andmitochondrial proteins as compared to the entire mouse proteome.S-nitrosylation sites in proteins in cellular membranes and nucleus wereunderrepresented. The underrepresentation of membrane proteins mayreflect methodological issues because the tissue homogenization methodwas not optimized for extraction of membrane proteins. S-nitrosylationof proteins also occurs in the nucleus, and the discovery of additionalsites in nuclear proteins reinforces the potential importance ofS-nitrosylation in signaling and transcriptional regulation (Kornberg etal. (2010) Nat. Cell Biol., 12:1094-1100). Twenty percent to 25% of theS-nitrosoproteome in the brain, kidney, liver, lung, and thymusconsisted of mitochondrial proteins, whereas 56% of the modifiedproteins were localized to mitochondria in the mouse heart. Themitochondrial proteomes were more than 70% dependent on eNOS activitywith the exception of the heart, where only 36% of the proteome requireseNOS-derived nitric oxide for S-nitrosylation. The lower dependency ofS-nitrosylation on eNOS activity in heart mitochondria indicates thepresence of another functional NOS isoform. Functional classification ofthe S-nitrosoproteome revealed key metabolic pathways in whichS-nitrosylated enzymes play a central role. It was found that asubstantial number of enzymes that regulate glycolysis, gluconeogenesis,pyruvate metabolism, tricarboxylic acid (TCA) cycle, oxidativephosphorylation, amino acid metabolism, ketone body formation, and fattyacid metabolic pathways were S-nitrosylated, most of which were notidentified as S-nitrosylated in eNOS^(−/−) mice. This finding isconsistent with reports indicating that the eNOS null mice have impairedmetabolic activity (Schild et al. (2008) Biochim. Biophys. Acta,1782:180-187; Mohan et al. (2008) Lab. Invest., 88:515-528). Therefore,it appears that protein S-nitrosylation provides the mechanistic linkthat couples eNOS activity and regulation of metabolism.

Regulation of the Enzymatic Activity of Very Long Chain Acyl-CoADehydrogenase by S-Nitrosylation

Clustering of proteins that participate in fatty acid metabolism wasapparent in this analysis. In the mouse liver, S-nitrosylated proteinsare clustered in a network that encompasses liver responses to thehormone leptin (Doulias et al. (2010) Proc. Natl. Acad. Sci.,107:16958-16963). Here, it was found that very long chain acyl-coenzymeA (CoA) dehydrogenase (VLCAD), which catalyzes the rate-limiting step inthe β-oxidation of fatty acids, was S-nitrosylated in wild-type mouseliver but not in the livers of mice lacking leptin (ob/ob) or eNOS^(−/−)mice (FIG. 1). The biological effect of S-nitrosylation on VLCADactivity and fatty acid metabolism in the liver, which is a majormetabolic organ, was investigated.

Leptin-deficient mice develop liver steatosis spontaneously under normalchow diet starting at 4 to 5 weeks of life that is characterized by adiminished rate of palmitate oxidation and accumulation of fat dropletsin the form of triglycerides within the hepatocytes (Brix et al. (2002)Mol. Genet. Metab., 75:219-226; de Oliveira et al. (2008) J. Am. Coll.Nutr., 27:299-305). The rate of ³H-labeled palmitoyl-CoA oxidation byliver homogenates in ob/ob mice was 47% of the rate in wild-type mice(FIG. 2A). This significant reduction in the rate of palmitoyl-CoAoxidation indicated a deficiency in mitochondrial β-oxidation of fattyacids. Based on observations that showed reversal of hepatic steatosisin ob/ob mice by delivery of S-nitroso-N-acetyl cysteine (de Oliveira etal. (2008) J. Am. Coll. Nutr., 27:299-305), 5-week-old ob/ob mice wereinjected intraperitoneally with S-nitrosoglutathione (GSNO). GSNO wasused to deliver nitric oxide equivalents because of the extensive use ofthis pharmacological agent in cellular models to modify proteins byS-nitrosylation despite limited cellular permeability (Hara et al.(2005) Nat. Cell Biol., 7:665-674; Chung et al. (2004) Science304:1328-1331). GSNO-injected ob/ob mice exhibited a similarpalmitoyl-CoA oxidation rate as wild-type mice and a significantlyincreased rate over phosphate-buffered saline (PBS)-injected ob/ob mice(FIG. 2A). Moreover, the restoration of palmitoyl-CoA oxidation inGSNO-injected ob/ob mice was associated with a significant reduction ofthe concentration of liver triglycerides. The liver triglycerideconcentration in ob/ob mice injected with PBS was significantly higherthan in wild-type mice, and treatment of ob/ob mice with GSNOsignificantly lowered the liver triglyceride concentration (FIG. 2B).The serum triglyceride concentrations were similar in wild-type mice,PBS-treated ob/ob mice, and GSNO-treated ob/ob mice (FIG. 2C). Therestoration of palmitoyl-CoA oxidation in GSNO-injected ob/ob mice wasalso corroborated by histological evaluation of liver slices, whichshowed fewer fat deposits in GSNO-injected than in PBS-injected ob/obmice (FIG. 2G).

MS/MS analysis localized the site of VLCAD modification to Cys238 inboth wild-type and GSNO-injected ob/ob mice (FIGS. 1A and 1B). It wasalso confirmed that VLCAD was not modified by S-nitrosylation ineNOS^(−/−) mice but was readily modified ex vivo at cysteine residueCys238 by treating liver homogenates with GSNO (FIGS. 2H and 2I).

The S-nitrosylation of VLCAD was associated with an increase in acyldehydrogenase-mediated oxidation of palmitoyl-CoA in GSNO-treated ob/obmouse liver (FIGS. 2D, 2J, and 2K). This increase was sensitive to UVphotolysis and was abolished by the inclusion of specific antibodiesagainst VLCAD (FIG. 2J). Analysis of Michaelis-Menten kinetics (FIG. 2D)revealed that the V_(max) of VLCAD was similar in wild-type,PBS-injected, and GSNO-injected ob/ob mice (FIG. 2E). However, theapparent Michaelis constant (K_(M)) value measured in homogenates fromPBS-injected ob/ob mice was more than fivefold higher than in those fromwild-type mice and GSNO-injected ob/ob mice (FIG. 2F). The K_(M) forVLCAD enzymatic activity also increased five-fold in eNOS^(−/−) liverhomogenates treated ex vivo with GSNO compared to untreated eNOS^(−/−)liver homogenate (FIG. 2H). Quantification of the abundance of VLCADprotein in liver homogenates and enriched mitochondria preparationsindicated no differences in abundance among wild-type, PBS-injected, andGSNO-injected ob/ob mice (FIGS. 3A and 3B). Furthermore, native gelelectrophoresis revealed equal abundance of VLCAD dimers in themitochondria fractions from all three groups of mice (FIG. 3A, upperpanel). Therefore, these data indicate that S-nitrosylation decreasesthe KM of VLCAD.

Quantification of the fraction of S-nitrosylated VLCAD indicated that inGSNO-treated mice, 25±3% of VLCAD molecules in liver wereS-nitrosylated. These findings confirm the MS/MS analysis showingS-nitrosylation of VLCAD in wild-type and GSNO-treated mouse liver butnot in PBS-treated ob/ob mouse liver (FIG. 3C). Because theconcentrations of VLCAD protein were similar in PBS- and GSNO-treatedob/ob mice and on average the steady-state abundance of S-nitrosylatedVLCAD was 25% of the total protein, S-nitrosylation can increase thecatalytic efficiency (k_(cat)/K_(M)) (Koshland, D. E. (2002) Bioorg.Chem., 30:211-213) of VLCAD 29-fold as compared to the unmodifiedprotein. This substantial increase in catalytic efficiency can provideefficient removal of fatty acids in ob/ob mouse liver.

Additional evidence for the functional consequences of S-nitrosylationof VLCAD was obtained in mouse hepatocytes transiently expressingwild-type VLCAD or a point mutant that could not be S-nitrosylated(C238A). GSNO treatment resulted in S-nitrosylation of 27±3% ofwild-type VLCAD (FIG. 4A) concomitant with a fivefold increase ofVLCAD-specific activity (FIG. 4B). Under the same experimentalconditions, the C238A VLCAD mutant was not S-nitrosylated, and its basalactivity did not change in response to GSNO treatment (FIGS. 4A and 4B).Upon removal of GSNO from the media and extensive washing, both theabundance of S-nitrosylated VLCAD and the enzymatic-specific activitydeclined over time (FIGS. 4A and 4B). These data indicate thatS-nitrosylation of VLCAD at Cys²³⁸ is necessary for the regulation ofits enzymatic activity, and this process is reversible, possiblycontrolled through denitrosylation.

Molecular dynamic simulation was used to gain insight into the effect ofS-nitrosylation on VLCAD structure. Quantum mechanics/molecularmechanics calculations was used initially to generate theS-nitrosocysteine residue at position 238 within the protein structureof VLCAD [ProteinDataBank (PDB) ID2UXW] (FIG. 5A). Examination of thelowest frequency normal modes, which indicate large global or collectivemotions of a protein, did not indicate that S-nitrosylation inducedlarge conformational changes. Examination of higher frequency modes toinvestigate smaller local motions that might occur near the binding siterevealed a difference in the movement of atoms in response toS-nitrosylation of Cys238 (FIG. 5B). Cys238 resides in a loop, and loopsgenerally represent flexible regions of a protein. Therefore, the dataindicate that S-nitrosylation enhances loop flexibility, resulting inincreased protein movement, which in turn facilitates substrate bindingand therefore lowers the K_(M) of VLCAD. Cys²³⁸ was ≥30 Å from thesubstrate binding site, indicating that longer-range motions caninfluence the ability of the enzyme to bind substrate.

Cysteine S-nitrosylation is a nitric oxide-derived posttranslationalmodification that modulates protein activity, protein-proteininteractions, and subcellular localization under physiological andpathological conditions (Stamler et al. (2001) Cell 106:675-683; Hess etal. (2005) Nat. Rev. Mol. Cell Biol., 6:150-166; Benhar et al. (2008)Science 320:1050-1054; Jaffrey et al. (2001) Nat. Cell Biol., 3:193-197;Kornberg et al. (2010) Nat. Cell Biol., 12:1094-1100; Matsushita et al.(2003) Cell 115:139-150; Mitchell et al. (2005) Nat. Chem. Biol.,1:154-158; Cho et al. (2009) Science 324:102-105; Mannick et al. (2001)J. Cell Biol., 154:1111-1116). Applying site-specific mapping ofS-nitrosocysteine residues in wild-type mouse tissue, widespreadmodification of proteins participating in metabolic pathways and asignificant localization of modified proteins in the mitochondria wasidentified. This selective localization is consistent with severalreports indicating functional roles for nitric oxide in mitochondrialbiology (Brown et al. (1994) FEBS Lett., 356:295-298; Kobzik et al.(1995) Biochem. Biophys. Res. Commun., 211:375-381; Nisoli et al. (2005)Science 310:314-317) and specifically for the heart, where proteinS-nitrosylation protects the heart from ischemia-reperfusion injury(Prime et al. (2009) Proc. Natl. Acad. Sci., 106:10764-10769; Lima etal. (2009) Proc. Natl. Acad. Sci., 106:6297-6302; Kohr et al. (2012)Circ. Res., 111:1308-1312). Delivery of nitric oxide donors specificallyto the heart increases the overall abundance of mitochondrialS-nitrosylated proteins and protects from ischemic injury (Prime et al.(2009) Proc. Natl. Acad. Sci., 106:10764-10769; Lima et al. (2009) Proc.Natl. Acad. Sci., 106:6297-6302; Kohr et al. (2012) Circ. Res.,111:1308-1312). Despite these important biological contributions ofnitric oxide in the functional regulation of mitochondria andmetabolism, the identification of S-nitrosylated mitochondrial proteinsis limited. The identification of specific sites of S-nitrosylation inmitochondrial proteins across six different mouse tissues provides asubstantial advance that facilitates mechanistic studies to uncovermolecular and biochemical pathways for nitric oxide-mediated regulationof bioenergetics and metabolism. Similar to other posttranslationalmodifications, S-nitrosylation is a dynamic process, and severalpathways that reverse this posttranslational modification have beendescribed (Benhar et al. (2008) Science 320:1050-1054). Therefore, thecurrent S-nitrosocysteine proteome may represent a portion of theendogenously modified proteins under normal physiological conditions.

To explore the biological role of S-nitrosylation in vivo, fatty acidmetabolism in the liver, a major metabolic organ in the body, wasstudied. Diet, adipose tissue, and de novo lipogenesis are the majorsources of fatty acids for the liver. Potential fates of fatty acids inthe liver include esterification to triglycerides, which can be storedor packaged with apolipoprotein B-100 for export as part of very lowdensity lipoprotein particles. Fatty acids can be also converted tophospholipids or transformed to acyl-carnitines for transport intomitochondria, where they undergo β-oxidation (Cohen et al. (2011)Science 332:1519-1523). Any process that increases the input ordecreases the output or metabolism of fatty acids potentiallycontributes to the development of liver steatosis (Cohen et al. (2011)Science 332:1519-1523). β-Oxidation is a four-step enzymatic cycle, andeach turn of the cycle shortens the length of the fatty acid by twocarbon atoms, which is critical for efficient use of fatty acids. VLCADcatalyzes the first step in β-oxidation, accounting for about 80% ofpalmitate dehydrogenase activity in human liver and nearly 70% ofpalmitate oxidation in mouse liver (Aoyama et al. (1995) J. Clin.Invest., 95:2465-2473; Djordjevic et al. (1994) Biochemistry33:4258-4264; Strauss et al. (1995) Proc. Natl. Acad. Sci.,92:10496-10500). VLCAD is a homodimer consisting of 67-kD subunits andis embedded in the inner mitochondria membrane (Aoyama et al. (1995) J.Clin. Invest., 95:2465-2473; Djordjevic et al. (1994) Biochemistry33:4258-4264; Strauss et al. (1995) Proc. Natl. Acad. Sci.,92:10496-10500). The enzymatic activity of VLCAD appears to be regulatedby protein abundance and phosphorylation of Ser⁵⁸⁶ (Kabuyama et al.(2010) Am. J. Physiol. Cell Physiol., 298:C107-C113). The data presentedhereinabove indicate that S-nitrosylation of VLCAD at Cys²³⁸ througheNOS-derived nitric oxide results in reversible activation of enzymaticactivity through conformational changes that alter the K_(M) of theenzyme and can substantially influence the in vivo β-oxidation of fattyacids. Overall, the global analysis of S-nitrosylated proteins in mousetissues revealed that this posttranslational modification can profoundlyinfluence cellular metabolic processes and mitochondria function.

Example 2

Nitric oxide (NO) regulates mitochondrial metabolism under normal andpathological conditions. One of the mechanisms by which nitric oxideachieve its regulatory effect is through the S-nitrosylation of cysteineresidues in proteins. As shown hereinabove, 25% of VLCAD molecules areS-nitrosylated at cysteine 238 in vivo (mouse models). S-nitrosylationlowers the apparent Km of VLCAD by 5-fold and improves the catalyticefficiency by 29-fold as compared to the unmodified enzyme. In an animalmodel that resembles features of the human disease (low VLCAD activity,low β-oxidation rate, hepatic accumulation of triglycerides),S-nitrosylation of VLCAD restored enzymatic activity, normalizedβ-oxidation rate and diminished the steatotic phenotype. Based on theseresults, S-nitrosylation of missense mutated human VLCAD will improveits catalytic efficiency and will positively affect the outcome of thedisease.

To test this conclusion, four human fibroblast lines carrying pathologicVLCAD mutations were used (Table 1). Cells were exposed to 100 μMN-acetylcysteine or S-nitroso-N-acetyl-cysteine (SNO-NAC) for 30 minutesand the S-nitrosylation status of VLCAD was determined byorganomercury-assisted capture and mass spectrometry as describedhereinabove. Using lysates from cell lines 1 and 2, it was determinedthat VLCAD was modified on cysteine 237 (human VLCAD has one fewer aminoacid) after SNO-NAC treatment. VLCAD enzymatic activity was assessed incell lysates by monitoring the ferrocenium reduction in the presence ofpalmitoyl-CoA as substrate for the enzyme. FIG. 6 presents theMichaelis-Menten traces for cell lines 1 and 2 showing thatS-nitrosylation of VLCAD has a prominent effect on the Km (14 fold loweras compared to the unmodified enzyme) and at a lesser extent on V_(max).

TABLE 1 Missense mutations on VLCAD. Each cell line had two mutations.Patient 1 G185S G295E Patient 2 P91Q G193R Patient 3 P89S A536fsX550Patient 4 N122D N112D

A detailed kinetics analysis was performed for the other mutants as wellas for the wild type VLCAD (Table 2). Low mitochondrial fatty acidoxidation (mFAO) capacity and VLCAD activity was documented inVLCAD-deficient fibroblasts treated with NAC as compared to controlcells. For Table 2, initial velocity (Vo) was determined in the linearpart of reaction curve by calculating the slope from time 0 to the timecorresponding to 5% loss of initial absorbance. For determination of Kmand Vmax, 10 concentrations of palmitoyl-CoA ranging from 0.015 to 2 mMwere used. mFAO rate was determined by quantifying ³H₂O released in theculture medium of cells incubated with ³H-palmitate. The table presentsthe average values of two different experiments. The two values do notdiffer by more than 10%.

In all cases the protein follows typical Michaelis-Menten kinetics. Allmutants had much lower basal activity than the corresponding activity ofthe wild type VLCAD. Their apparent Michaelis constant (Km) was7-14-fold higher than the Km of the wild type protein whereas theirapparent Vmax was either 15-50% lower or double as compared to the wildtype VLCAD. Importantly, S-nitrosylation of mutated VLCAD lowered theapparent Km by a factor ranging from 7 to 14 fold (on average 11 folddecrease). In addition, S-nitrosylation of mutated VLCAD slightlyreduced the Vmax. Overall, the data indicates that S-nitrosylation ofVLCAD on cysteine 237 corrects the enzymatic deficiency of mutated VLCADby lowering the Km. In other words, treatment withS-nitroso-N-acetylcysteine (SNAC) increased VLCAD enzymatic activityreflected by normal values of apparent K_(M), and restored mFAO capacityin VLCAD deficient cells.

TABLE 2 Kinetics parameters of mutant and wild-type VLCAD. NAC SNO-NACmFAO mFAO V_(max) K_(M) (nmol/ V_(max) K_(M) (nmol/ (μU/mg) (mM) mg/h)(μU/mg) (mM) mg/h) Patient 1 125 1.14 0.6  83 0.09 3.9 (G185S/G295E)Patient 2 155 1.52 0.4 119 0.11 4.7 (P91Q/G193R) Patient 3 239 1.07 0.8160 0.17 4.9 (P89S/A536fsX550) Patient 4 604 1.85 0.5 343 0.16 4.1(N122D/N122D) Healthy donor 269 0.13 2.6 259 0.08 3.9

Furthermore, a mercury resin assisted capture (MRC) technology followedby mass spectrometry based detection was used to confirm that SNO-NACtreatment results in the S-nitrosylation of cysteine 237. Specifically,the MS/MS spectrum revealed the presence of the double charged peptideTSAVPSC₂₃₇GKYYTLNGSK, corresponding to sequence 231-247 of human VLCAD,only in cell homogenates from SNAC-treated VLCAD-deficient cells.Notably, no other modification of VLCAD was detected. These datademonstrate that S-nitrosylation of VLCAD increased catalytic efficiencyin the presence of other mutations and show that protein S-nitrosylationcorrects VCLAD enzymatic deficiency.

While certain of the preferred embodiments of the present invention havebeen described and specifically exemplified above, it is not intendedthat the invention be limited to such embodiments. Various modificationsmay be made thereto without departing from the scope and spirit of thepresent invention, as set forth in the following claims.

What is claimed is:
 1. A method for treating a fatty acid oxidationdisorder in a subject, said method comprising administeringS-nitroso-N-acetyl-cysteine (SNO-NAC) or a pharmaceutically acceptablesalt thereof to said subject, wherein said fatty acid oxidation disorderis very long-chain acyl-coenzyme A dehydrogenase deficiency (VLCADD). 2.The method of claim 1, wherein the subject has a missense or nullmutation within very long-chain acyl-coenzyme A dehydrogenase (VLCAD).3. The method of claim 2, wherein the subject has a missense mutationwithin very long-chain acyl-coenzyme A dehydrogenase (VLCAD).
 4. Themethod of claim 1, further comprising diagnosing a fatty acid oxidationdisorder in said subject prior to administration of said S-nitrosylatingagent.
 5. The method of claim 4, wherein said diagnosis comprises: a)obtaining a biological sample from said subject; b) determining theenzymatic activity of the very long-chain acyl-coenzyme A dehydrogenase(VLCAD) in said sample; and c) comparing the amount of VLCAD enzymaticactivity determined in step b) to the amount of VLCAD enzymatic activityin a corresponding biological sample from a healthy subject, wherein adecrease in the VLCAD enzymatic activity in the biological sample fromthe subject compared to the healthy subject is indicative of a fattyacid oxidation disorder in said subject.
 6. The method of claim 4,wherein the diagnosis comprises determining the presence of a mutationin the very long-chain acyl-coenzyme A dehydrogenase (VLCAD) encodingnucleic acid molecule in a biological sample obtained from said subject,wherein the presence of a mutation in the VLCAD encoding nucleic acidmolecule is indicative of a fatty acid oxidation disorder in saidsubject.
 7. The method of claim 1 further comprising the administrationof at least one other therapeutic agent for the treatment of the fattyacid oxidation disorder.
 8. The method of claim 7, wherein said othertherapeutic agent is triheptanoin or bezafibrate.